EXECUTIVE SUMMARY - huESC HTS2 - human Toxic stress effects in 1st trimester pregnancy leads to $10s of billions of US health care costs/year and is a significant part of the $2.5-4B/year US Developmental and Reproductive Technology (DART industry. Current DART techniques use slow, expensive pregnant rodent tests, or tests of embryonic stem cells (ESCs) that are cultured to lose ?stemness? and differentiate into organ tissues. As differentiated organ tissues, our competitors DART high throughput screens (HTSs) test only metabolic or cytotoxic effects of compounds on cell survival and function. ReproStress Inc. exposes undifferentiated ESCs to stress and test for organismal survival outcomes of developing embryos. We have shown, and developed ESC HTSs to test, stress-forced changes in stemness loss and abnormal differentiation to 1st lineage while suppressing later lineages. These outcomes should assess organismal risks for stunting, teratogenesis and miscarriage. Our HTSs should be able to reduce, refine, and replace many organismal DART assays now done in pregnant females. Reproductive Stress 3M has invented HTSs that test toxicants on transgenic reporter ESCs cultured as stem cells that accurately report stunted ESC quality and growth. These HTSs show stress-forced stem cell stunting, differentiation and depletion that predicts drug exposures that are embryotoxic and unsafe for development. ReproStress? patented stemness loss ESC HTS(1) identifies nonembryotoxic drugs such as Penicillin, weak or strong embryotoxic drugs such as Salicylate or Methotrexate, respectively. In 2019, we published a paper and converted the provisional patent for mouse (mu)ESC HTS2 which reports 1st lineage increase. Commercial customers and government regulators want conversion of mouse to human (hu)ESC HTS. Although rare in huESC, we found 2 huESC lines in the GEO transcriptomic databases, NIH-approved H1 and H9 lines, that do differentiate 1st lineage. In Aim 1 we will confirm that H1 and/or H9 huESCs produce 1st lineage during normal differentiation, and more 1st lineage marker/cell during stress-forced differentiation. In Aim 1 we will make transgenic huESC with H1 or H9 with two improvements over muESC HTS2; 1) we will change high background fluorescent reporters to low background, rapid turnover luminescent reporters. To drive luminescence reporters, our current R41 transcriptomic data suggested stronger promoters for 1st lineage exist by two criteria. Stronger promoters should enable a higher resolution, more reproducible assay and we will test patented Pdgfra promoter and a second, stronger 1st lineage reporter for both criteria. In Aim 1, we increase the validating drug set size to show the in vitro HTSs? reproducibility and predictive nature for in vivo toxicological outcomes after gestational exposures of the same compounds. In Aim 2, we analyze single cell global transcriptomic marker to corroborate the luminescence marker itself and support the theory that stress forces differentiation unique to 1st lineage and decreases later ones; leads to teratogenesis. These aims will lead to a Phase 2 grant to test compounds from the large 10k NIEHS toxicant set, leading to regulatory approval and sales to DevTox testing for commercial customers.